Telomeres protect genome stability by preventing chromosomes from inappropriate fusions, degradation, and recombination. Failure of telomere protection results in genome instability that leads to diseases such as cancer and premature aging. Telomeres achieve their protection function through the formation of distinct DNA-protein structures termed t-loops. Disruption of t-loop leads to exposure of telomere ends, triggering DNA damage response and inducing cellular senescence or apoptosis. Therefore, inducing telomere dysfunction is a promising approach for cancer therapy. One component of t-loop, the single-stranded G-rich overhang (G- overhang), has multiple vital roles in regulating telomere homeostasis: (1) it is essential for the formation of the t-loop structure, (2) it is required for telomerase binding to telomeres, (3) it is needed for lengthening telomeres in certain cancer cells using telomerase-independent pathway, and (4) the process for generating G-overhang contributes to telomere shortening. Thus, deciphering the mechanism for G-overhang generation is crucial for understanding telomere maintenance in both telomerase-expressing and non-expressing cancer cells. However, the molecular mechanism for generating G-overhangs is poorly understood in human cells. Lack of such knowledge is a significant barrier for devising effective therapeutic strategies for cancer cells. Our preliminary studies indicate that telomeric G-overhangs are generated by multiple tightly regulated steps during the cell cycle. We have also revealed the unidentified role of cyclin-dependent kinase 1 (CDK1) in controlling G-overhang generation and in maintaining functional telomeres in cancer cells. The central hypothesis is that G-overhangs are generated by multiple tightly regulated steps during the cell cycle under the control of CDK1. The specific aims are: (1) Define the molecular events responsible for G-overhang generation at leading and lagging daughter telomeres during the cell cycle. (2) Determine the molecular pathway by which CDK1 regulates G-overhang generation. We will combine biochemical and molecular approaches to determine the pathway by which CDK1 regulates telomere maintenance. The findings from these studies will advance our understanding in the mechanism for maintaining telomere integrity and genome stability, aid us in understanding telomere maintenance mechanism in cancer cells and in developing new regimens for cancer therapy. PUBLIC HEALTH RELEVANCE: Maintenance of telomere integrity is essential for the unlimited growth of cancer cells. This proposal aims to yield new insights into telomere maintenance in cancer cells, cell growth control and oncogenesis. It may lead to the development of new anti-cancer therapy.